The MHC II ligandome [Elektronische Ressource] : mass spectrometric applications in immunology = Das MHC-II-Ligandom / vorgelegt von Jörn Dengjel

The MHC II Ligandome
Mass Spectrometric Applications in Immunology



Das MHC II Ligandom
Massenspektrometrische Anwendungen
in der Immunologie





DISSERTATION

der Fakultät für Chemie und Pharmazie
der Eberhard-Karls-Universität Tübingen

zur Erlangung des Grades eines Doktors
der Naturwissenschaften


2005

vorgelegt von


Jörn Dengjel

II


































III






























Tag der mündlichen Prüfung: 24.01.2005

Dekan: Prof. Dr. S. Laufer
1. Berichterstatter: Prof. Dr. S. Stevanovi ć
2. Berichterstatter: Prof. Dr. H.-G. Rammensee IV


































V

Preface

Some of the chapters of this thesis are parts of publications. At the beginning of
each chapter it is indicated which experiments where done by the author of the
thesis, which persons contributed to the publication, and in which journals the work
was published. VI





























Table of Contents 1

Table of Contents
TABLE OF CONTENTS 1
1 INTRODUCTION 3
1.1 The immune system 3
1.1.1 Major histocompatibility complex molecules and antigen processing 4
1.1.2 T lymphocytes and anti-tumor immunotherapy 6
1.2 Mass spectrometry 9
1.2.1 MALDI-TOF mass spectrometry 10
1.2.2 ESI-quadrupole-TOF mass spectrometry 11
1.3 Aims of the thesis 16
1.4 References 17
2 RESULTS AND DISCUSSION 27
2.1 Autophagy promotes MHC-II presentation of peptides from
intracellular source proteins 27
2.1.1 Summary 27
2.1.2 Introduction 28
2.1.3 Materials and Methods 29
2.1.4 Results 33
2.1.5 Discussion 57
2.1.6 References 60
2.2 Glycan side chains on naturally presented MHC class II ligands 66
2.2.1 Summary 66
2.2.2 Introduction 66
2.2.3 Materials and Methods 67
2.2.4 Results and Discussion 69
2.2.5 References 74 2 Table of Contents

2.3 Identification of a naturally processed cyclin D1 T-helper epitope by a
novel combination of HLA class II targeting and differential mass
spectrometry 78
2.3.1 Summary 78
2.3.2 Introduction 79
2.3.3 Materials and Methods 80
2.3.4 Results 85
2.3.5 Discussion 91
2.3.6 References 92
3 SUMMARY 98
4 ABBREVIATIONS 100
5 ACKNOWLEDGEMENT 101
6 ACADEMIC TEACHERS 103
7 PUBLICATIONS 104
8 SCHOLARSHIPS 105
9 CURRICULUM VITAE 106











Introduction 3

1 Introduction


1.1 The immune system

Although our body is daily confronted with possible invasions of pathogens, severe
infections occur only seldom, due to our highly efficient immune system. It is
responsible for host defense against infectious agents. With the help of a complex
system of humoral and cellular defense mechanisms it is able to discriminate
between self and non-self protecting our body against pathogens. The immune
system consists of all organs, tissues, cells and molecules involved in host
defense. Immunocompetent organs can be broadly divided into central and
peripheral. In the central organs immunocompetent white blood cells – leukocytes
– are generated and major parts of their development take place: the bone
marrow, where generation of leukocyte progenitors from pluripotent hematopoetic
stem cells occurs and B lymphocytes mature, and the thymus, the organ of T
lymphocyte maturation. The most important peripheral organs are the spleen, the
lymph nodes and the mucosal lymphatic tissues. Here, lymphocytes are
maintained and meet possible antigens.
Defense mechanisms of the immune system, the so called immune response, are
based on two different but interacting principles, innate and adaptive immunity.
Innate immunity provides a first line of defense against many common pathogens.
It evolved before adaptive immunity and plays a crucial role in controlling
infections in the first four to seven days, the time needed before an initial adaptive
immune response can take effect. Part of an innate immune response are: the
complement system, inflammatory cells – macrophages and neutrophils –, natural
killer cells (NK cells), γ: δ T cells and B-1 B cells. The components of innate
immunity are constitutively present in our body and do not generate an
immunological memory.
Adaptive immunity is based on clonal selection of antigen-specific effector
lymphocytes and on generation of memory cells that are able to prevent re-
infection with the same pathogen. An adaptive immune response, also known as
acquired immune response, is mainly made up by three different cell types: 4 Introduction

professional antigen-presenting cells (APC), B lymphocytes and T lymphocytes.
To raise such a response at least two of the above mentioned cell types have to
participate. Adaptive immunity distinguishes humoral, or B cell-mediated, and
cellular, or T cell-mediated, immunity. Humoral immunity is mainly based on
antibodies (Ab) secreted by B cells which have to get activated by antigen-specific
T helper cells (TH2), and is directed against extracellular pathogens and toxins.
Cellular immunity, in general directed against intracellular pathogens or for
example tumor cells, is based on cytotoxic T lymphocytes (CTL) and inflammatory
TH1 cells. To become activated, CTL have to interact with APC and TH1 cells.
Coordination and regulation of a specific immune response are maintained
through subpopulations of T cells that either activate or inhibit other immune
competent cells.


1.1.1 Major histocompatibility complex molecules and antigen processing

As already mentioned, the immune system is able to distinguish between self and
non-self through humoral and cellular defense mechanisms, respectively. B cell
receptors and Ab bind three dimensional, native structures, whereby nearly every
chemical substance can be recognized by Ab. T cells, on the other hand, are more
limited. Via their T cell receptor (TCR), they recognize parts of processed proteins,
peptides, which have to be presented on special receptors, the major
histocompatibility complex (MHC) molecules, on cell surfaces. The two different
subsets of T cells, CD8+ and CD4+ T cells, recognize two different types of MHC
molecules, MHC class I and MHC class II, respectively. The MHC is the most
polymorphic gene cluster in humans and is located on chromosome six. MHC
molecules, in humans called human leukocyte antigens (HLA), are a family of
highly polymorphic glycoproteins. MHC I molecules are heterodimers formed of a
heavy chain, in humans HLA-A, -B, -C, and of a noncovalently bound light chain,
β2-microglobulin ( β m). In contrast, MHC II molecules, in humans HLA-DR, -DQ, -2
DP, are made of two heavy chains, α and β.
In the classical view, MHC class I molecules present peptides from endogenously
synthesized proteins on cell surfaces of the vast majority of cells, allowing